Supplementary data
Profiling and identification of the metabolites of ginsenoside Ro in
rat faeces and urine after oral administration
Jia Wang, Peihe Zheng, Yufang Wang, Yanzhu Zhu, Hua Zhou, Shengyuan Xiao, Yingping Wang
Preparation of silphioside F and glucosyl oleanolate
1. Silphioside F (calenduloside E)
Silphioside F was isolated from the acidic hydrolysate of ginsenoside Ro. Briefly, approximately 50 mg
ginsenoside Ro was dissolved in 5 ml of oxalic acid solution (pH=1.0) in a flask. The flask was heated in a bath of
boiling water for 2h. Then the flask was taken out and cooled to ambient temperature in tape water before the
acidic solution was neutralized using ammonium solution (10%, w/v). The solution was filtered after the same
volume of methanol was added and was finally injected into the HPLC for separation (Fig. S1).
Figure S1 Chromatogram for silphioside F preparative isolation (upper) and total ion chromatogram of HPLC ESI
MS analysis of the prepared silphioside F (lower)
The separation was performed using a HPLC over a semi preparative column (VARIAN Polaris C18-A, 10mm ×
250mm, 10m). The mobile phase consisted of pure water (A) and acetonitrile (B). A gradient elution was used as
follows: 15% B at the first 3 min, then a linear gradient of 15 – 50% B for 3 – 20 min, 50 – 95% B for 20 – 23 min,
and this composition was maintained for 23-28 min. The flow rate was 4 ml/min. The chromatogram was recorded
at 203 nm. The injection volume was 300 l in each performance. An eluant from 19.0 – 19.5 min was collected,
concentrated and lyophilized. The residue was recrystallized for usage (Fig. S1).
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2. Glucosyl oleanolate
Glucosyl oleanolate was isolated form the extract of rat faeces collected after oral administration of ginsenoside
Ro. Briefly, the faeces extract was absorbed on silicon gel and dried. The gel was then extracted with petroleum
and ethyl acetate successively. The ethyl acetate eluant was dried under a flow of nitrogen. The residue was
dissolved using water (containing 10% methanol, v/v). And then the aqueous solution was load onto a reversed
phase SPE column. The column was washed with water (containing 10% methanol, v/v) and methanol
successively. The methanol eluant was then concentrated, filtered and injected into an HPLC for separation.
The separation was carried out using an Agilent HPLC (1200 series) on a Agilent TC-C18 (4.6mm × 250mm，5m)
column. The mobile phase consisted of pure water (A) and acetonitile (B). A gradient elution was used as follows:
20% B at the first 4 min, and then a linear gradient of 20 – 29% B for 4 – 7 min, 29 – 40% B for 7 – 10 min, this
composition was maintained for 10 – 12 min, then 40 – 53.3 % B for 12 - 17 min, 53.3 – 56.6 % B for 17 – 22
min, 56.6 – 95% B for 22 – 30 min, this composition was maintained for 30 – 35 min. The flow rate was 1.0
ml/min. The wavelength of the detector was set as 203nm. The injection volume was 40 l. the peak at about 29.0
min was collected. The eluant was combined and dried under reduced pressure. Then the residue was dissolved in
water (containing 10% methanol, v/v) before loaded into an SPE column. The column was washed with water
(containing 10% methanol, v/v) and methanol successively. The methanol eluant was then concentrated and
purified using a sephadex LH20 open column chromatography (10.0 × 200 mm) and was finally diluted using
pure water and then lyophilized for use.
Figure S2 Chromatogram for glucosyl oleanolate preparative isolation (203nm, upper) and total ion chromatogram
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of HPLC ESI MS analysis of the prepared glucosyl oleanolate (lower)
The purity of prepared silphioside F and glucosyl oleanolate were determined using HPLC UV/ESI MS (Fig. S1
and Fig. S2). Their purities were calculated using the normalization peak areas. The identification of silphioside F
and glucosyl oleanolate was based on the structural relationship between the hydrolysate and the reactant (Fig. S4)
and the MS/MS data. Both silphioside F and glucosyl oleanolate were detected in the hydrolysates of ginsenoside
Ro. The identification of glucosyl oleanolate in the faeces was based on the comparison of it retention time on the
reversed phase HPLC column and MS/MS data with that detected in the hydrolysate (Fig. S3).
Figure S3 Total chromatogram of acidic hydrolysate of ginsenoside Ro and Extracted ion chromatogram at m/z
617 in negative mode HPLC ESI MS
Figure S4 Acidic hydrolysis reaction of ginsenoside Ro
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Fig S5 Negative CID spectra of ginsenoside Ro and its metabolites (from top to down: Ro, M2, M1, M4, M3 and
M7)
Fig S6A Positive all-scan MS spectra of ginsenoside Ro and its metabolites (from top to down: Ro, M2, M1, M4,
and M7)
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Fig S6B Positive CID spectra of ginsenoside Ro and its metabolites (from top to down: Ro, M2, M1, M3 and M7)
Fig S6C Extensive CID spectra of dehydrated fragments of ginsenoside Ro and its metabolites (from top to down:
Ro, M2, M1, M4, M3 and M7)
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Fig S6D Extensive CID spectra of dehydrated fragments of ginsenoside Ro and its metabolites (zoom in to shown
fragment at m/z 249, from top to down: Ro, M2, M1, M4, M3 and M7)
Fig S7A Negative ESI MS spectra of metabolite M5 and M9: A, All-scan MS spectrum of metabolite M9; B, CID
spectrum of deprotonated metabolite M9 (m/z 779); C, All-scan MS spectrum of metabolite M5; D, CID
spectrum of deprotonated metabolite M5 (m/z 779); E, CID spectrum acetate adduct anion of metabolite M5 (m/z
839); All CIDs were performed in auto MS/MS mode
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Fig S7B MS spectrum of metabolite M9 obtained under positive mode (from top to down, all-scan MS spectrum,
CID spectrum of precursor at m/z 798, CID spectrum of precursor at m/z 803 and CID spectrum of precursor at
m/z 439)
Fig S7C MS spectrum of metabolite M5 obtained under positive mode (from top to down, all-scan MS spectrum,
CID spectrum of precursor at m/z 798 and CID spectrum of precursor at m/z 803)
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Fig S8A MS spectrum of metabolite M10 obtained under positive mode (from top to down, all-scan MS spectrum,
CID spectrum of precursor at m/z 455 and CID spectrum of precursor at m/z 409)
Fig S8B Positive CID spectra of metabolite M10 and M13: A, CID spectrum of protonated oleanonic acid (M10)
(m/z 455); B, CID spectrum of protonated metabolite M13 (m/z 471); All CIDs were performed in auto MS/MS
mode
Fig S8C MS spectrum of metabolite M8 obtained under negative mode (upper, All-scan spectrum, lower, CID
spectrum of precursor at m/z 675)
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Fig S8D MS spectrum of metabolite M8 obtained under positive mode (from top to down: all-scan MS spectrum;
CID spectrum of adduct ion at m/z 634 [M+NH4+]; CID spectrum of protonated aglycon; and CID spectrum of
decarboxylated aglycon)
Fig S9A CID spectrum of Metabolite M12, M14, M11 and oleanolic acid obtained under negative mode
(From top to down: M12, M14, M11 and oleanolic acid)
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Fig S9B MS spectrum of Metabolite M12 obtained under positive mode
(From top to down: all-scan MS spectrum; CID spectrum of protonated molecular ion at m/z 457; CID spectrum
of dehydrated fragment at m/z 439)
Fig S9C Online UV of oleanolic acid (red) and metabolite M12 (blue)
Fig S9D MS spectrum of Metabolite M14 obtained under positive mode
(From top to down: all-scan MS spectrum; CID spectrum of protonated molecular ion at m/z 457; CID spectrum
of dehydrated ion at m/z 439)
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Fig S9E MS spectrum of Metabolite M11 obtained under positive mode
(Upper, all-scan MS spectrum; down, CID spectrum of decarboxylated ion at m/z 421)
Fig S10 CID dissociation pattern of metabolite M14 (upper) and M11 (lower)
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Table S1 calibration curves, LOD and LOQ for six ginsenosides
Metabolite
Linearity
r
Dynamic range
(pg per inj)
LOD
(pg per inj)
LOQ
(pg per inj)
ginsenoside Ro
chikusetsusaponin Iva
zingibrosideR1
silphioside F
β-D-glucopyranosyl oleanolate
oleanolic acid
y = 2.28189 x + 1.98166
y = 5.36815 x - 21.7165
y = 1.0161 x - 4.78364
y = 1.83099 x - 3.74025
y = 11906.9 x + 535.352
y = 5.51195 x - 3.839
0.9985
0.9989
0.9981
0.9991
0.9951
0.9992
10.2-2040.0
12.4-2480.0
11.5-2300.0
5.0-1000.0
41.0-8200.0
12.3-2460.0
2.1
2.5
3.1
1.7
4.2
0.7
6.9
8.2
10.2
4.2
14.6
2.2
Table S2 Recoveries of six ginsenosides in urine and faeces at different concentrations
compounds
urine
faeces
Recovery(%) RSD(%)
Recovery(%) RSD(%)
ginsenoside Ro
low
95.56
9.74
103.67
9.07
middle 92.73
3.73
93.93
8.04
high
86.71
1.71
90.07
2.91
chikusetsusaponin Iva
low
91.52
7.68
98.18
9.80
middle 89.51
5.04
92.05
9.87
high
84.10
5.34
88.13
6.47
zingibrosideR1
low
100.84
11.75
102.20
9.25
middle 94.70
5.57
98.14
7.64
high
89.16
5.14
90.32
3.36
silphioside F
low
99.49
5.99
103.70
13.09
middle 106.98
3.16
94.56
8.02
high
92.05
9.13
93.33
2.88
oleanolic acid
low
98.30
4.99
98.35
8.78
middle 88.41
5.52
87.88
5.63
high
89.17
5.19
105.20
2.65
β-D- glucopyranosyl oleanolate low
92.32
7.46
101.45
3.56
middle 90.13
5.67
97.69
6.47
high
95.78
3.64
95.21
8.86
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